**3. Role of WWTP in dissemination of ARG**

Freshwater resources are too limited and meeting the needs for water is challenging in the last decades as urban water shortages increase [36–38]. Based on the united nations world water development report of UNESCO in 2015, up to 70% of the fresh water, we take from rivers and groundwater is devoted to irrigation [38]. The predicted increase in the global human population to 9.7 billion in 2050 will lead to an increase in water requirement for agricultural and food production purposes [39]. Hence, the reuse of treated wastewater in agriculture seems to be a practical solution for water shortage [35]. In addition, it can help freshwater ecosystems by reducing the discharge of effluent from wastewater treatment plants (WWTPs) and preventing eutrophication and algal blooms [35].

Treated urban wastewater contains organic substances (e.g., antibiotics) and inorganic matters including pathogens, ARB and ARGs [40]. The reuse of treated wastewater may result in contamination of the environment and spread of ARB and ARGs and trigger public health concerns. One of the applications of treated wastewater is irrigation which is encouraged by governments and official organization especially because of water shortage and poverty in developing countries and urban areas [36, 41–43].

Zhang et al. [43] studied the contribution of wastewater treatment to the antibiotic resistance development of *Acinetobacter* spp. that are found in many environments, including water, soil, sewage, and food. In this study, *Acinetobacter* spp. isolates from five different sites including raw influent, second effluent, and final effluent of WWTP and upstream and downstream of the treated wastewater discharge point. This study determined the antibiotic susceptibility phenotypes using the disc-diffusion method for eight antibiotics that includes amoxicillin, chloramphenicol, ciprofloxacin, colistin, gentamicin, rifampin, sulfisoxazole, and trimethoprim. This research concluded that conventional biological treatment process in WWTPs increases the ARB population [43].

Another comprehensive study detected 140 plasmid-borne ARGs of the WWTP using polymerase chain reaction (PCR) method [44]. In this study, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Samples were collected from activated sludge and the final effluents of the WWTP. The methods included (a) isolation of plasmids from resistant bacteria, (b) selection of target reference ARGs and design of PCR primers, (c) PCR and amplicon detection, (d) sequencing and analysis of selected resistance-gene-specific amplicons. Based on the results of this study, bacteria of the WWTP share a mobile pool of ARGs that result in genetic exchange between clinical and WWTP bacteria. The final effluent of WWTP also contained ARB that confirms that the WWTP's final effluents are disseminating antibiotic resistance in the environment [44].

Recently, Zhang et al. studied both cell-free DNA and cell-associated DNA as a source for ARGs that are related to WWTPs. The cell-free DNA is extracellular DNA that can transform into other cells, and cell-associated DNA is intercellular DNA. The 0.22 μm filter intercepts intercellular DNA and extracellular DNA (filtrates contains the extracellular DNA). In this research, four ARGs (*sul(2), tet(C), blaPSE-1,* and *erm(B)*) as cell-associated and cell-free fractions were studied. The cell-associated DNA and cell-free DNA were independently extracted and ARGs copy numbers were quantified using qPCR. Based on the results of this study, cell-associated ARGs were more than ARGs fraction in the raw wastewater, however, after biological treatment, sludge settling, membrane filtration, and disinfection, cell-associated ARGs were removed considerably and cell-free ARGs removal was much lower. Therefore, the abundance ratio of cell-free ARGs to cell-associated ARGs increased. Cell-free ARGs are important pollutants from WWTPs which are potential risks to the effluent receiving environments [45].

**3. Role of WWTP in dissemination of ARG**

82 Antimicrobial Resistance - A Global Threat

**Resistance mechanism Metal ions Antibiotics**

Drug and metal sequestration Zn, Cd, Cu CouA

**Table 2.** Shared characteristics of antibiotic and metal resistance systems [21].

Drug and metal alterations As, Hg ß-lactams, Chlor Drug and metal efflux Cu, Co, Zn, Cd, Ni, As Tet, Chlor, ß-lactams Alteration of cellular targets Hg, Zn, Cu Cip, ß-lactams, Trim, Rif

Reduction in permeability As, Cu, Zn, Mn, Co, Ag Cip, Tet, Chlor, ß-lactams

and preventing eutrophication and algal blooms [35].

developing countries and urban areas [36, 41–43].

WWTPs increases the ARB population [43].

Freshwater resources are too limited and meeting the needs for water is challenging in the last decades as urban water shortages increase [36–38]. Based on the united nations world water development report of UNESCO in 2015, up to 70% of the fresh water, we take from rivers and groundwater is devoted to irrigation [38]. The predicted increase in the global human population to 9.7 billion in 2050 will lead to an increase in water requirement for agricultural and food production purposes [39]. Hence, the reuse of treated wastewater in agriculture seems to be a practical solution for water shortage [35]. In addition, it can help freshwater ecosystems by reducing the discharge of effluent from wastewater treatment plants (WWTPs)

Treated urban wastewater contains organic substances (e.g., antibiotics) and inorganic matters including pathogens, ARB and ARGs [40]. The reuse of treated wastewater may result in contamination of the environment and spread of ARB and ARGs and trigger public health concerns. One of the applications of treated wastewater is irrigation which is encouraged by governments and official organization especially because of water shortage and poverty in

Zhang et al. [43] studied the contribution of wastewater treatment to the antibiotic resistance development of *Acinetobacter* spp. that are found in many environments, including water, soil, sewage, and food. In this study, *Acinetobacter* spp. isolates from five different sites including raw influent, second effluent, and final effluent of WWTP and upstream and downstream of the treated wastewater discharge point. This study determined the antibiotic susceptibility phenotypes using the disc-diffusion method for eight antibiotics that includes amoxicillin, chloramphenicol, ciprofloxacin, colistin, gentamicin, rifampin, sulfisoxazole, and trimethoprim. This research concluded that conventional biological treatment process in

Another comprehensive study detected 140 plasmid-borne ARGs of the WWTP using polymerase chain reaction (PCR) method [44]. In this study, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Samples were collected from activated sludge and the final effluents of the WWTP. The methods included (a) isolation of plasmids from resistant bacteria, (b) selection of target reference ARGs and design of PCR primers, (c) PCR Munir and Xagoraraki [16] quantified 18 biosolids samples from seven WWTPs using qPCR methods. The mean concentrations of *tet(W), tet(O),* and *sul(1)* in all samples of biosolids were 9.53 × 108 , 3.15 × 108 , and 6.04 × 108 , respectively. Lime-stabilized biosolids had considerably (p < 0.05) lower concentrations of ARGs compared with other biosolids treatment methods. In this study, two different sites were observed for 4 months to investigate levels of ARGs (*tet(W), tet(O),* and *sul(1)*) in soils fertilized with manure or biosolids. The concentration of ARGs was higher in manure than biosolids, but surprisingly, the results showed no notable change in the concentration of ARGs in the samples of soil, since genetic diversity and natural characteristics of background soil minimized the effect of biosolids [16].

In a recent study by D'Angelo [46] on the potential risks of the presence of antibiotic in biosolid amendments, sorption and desorption of tetracycline were indicated. Their research was on four types of amendments including biosolids, poultry manure, wood chip litter, and rice hull litter at different temperatures. The sorption and desorption equilibrium constant in municipal biosolids was 20 times higher than other amendments since the concentration of bound Al3+ and Fe3+ is higher in municipal biosolids. Results showed that the sorption of tetracycline was significantly increased after treatment with alum and treatment of amendments would effectively reduce antibiotic diffusion rates [46].

The effect of treated urban wastewater irrigation on fungi diversity and soil microbial activities was studied by Alguacil and her team, in Spain. Based on this study, fungi diversity was higher in soil irrigated by fresh water, but microbial activities of soil irrigated by wastewater were much more than the soil irrigated by fresh water. Hence, wastewater not only had no negative effects on crop vitality but also developed fertility of the soil. Microbiological components are biotic factors of soil that might be altered by the increase of soil microbial biomass due to wastewater irrigation [47].

As mentioned before, WWTPs are known as sources of antibiotic resistance. Auerbach et al. [14] studied two activated sludge wastewater treatment plants and two freshwater lakes for the presence of 10 tetracycline resistance genes. Qualitative PCR and quantitative PCR methods were used to detect tetracycline resistance genes and quantify the number of tetracycline resistance gene copies per volume of sample, respectively. Their results showed that both WWTPs contain more diverse types of tetracycline resistant genes than the background natural lake water samples. They revealed that the WWTPs are a source of ARGs dissemination. *tetQ* and *tetG* in the treatment processes were attenuated, however, the UV disinfection did not reduce the ARGs [14].

In another study, Munir et al. [56] investigated the occurrence and distribution of ARGs including *sul*(1), *tet*(W), and *tet*(O) and their associated bacteria in the effluent of five WWTPs to assess the efficiency of different processes. ARGs and ARB removal ranged 2.37-log to 4.56-log in activated sludge, oxidative ditch and rotatory biological contactors and 2.57-log

Development of Antibiotic Resistance in Wastewater Treatment Plants

http://dx.doi.org/10.5772/intechopen.81538

85

Removal of antibiotics including sulfamethazine, sulfamethoxazole, trimethoprim, and lincomycin had been studied in five different WWTPs using aerobic/anaerobic treatment methods [57]. The results of this study showed the range of −11.2% to 69.0% efficiency for different pharmaceutical compounds including sulfamethazine, sulfamethoxazole, trimethoprim, and lincomycin. The negative removal efficiency belonged to lincomycin and because of its high

To sum it up, aerobic reactors alone are not very effective and biological treatment methods can remove antibiotics, ARB, and ARGs successfully if anaerobic and aerobic reactors operate in sequence. Despite the fact that anaerobic treatment is energy efficient and has high performance, aerobic treatment is more common in municipal WWTPs. Anaerobic treatments are often used to treat wastewater that contains high loads of organic matter like industrial wastewater and needs warm temperature (35°C). Activated sludge, which is an aerobic treatment, is studied in this project and the results will help to advance the efficiency of activated

Some studies aimed to remove ARGs in raw domestic wastewater by *constructed wetlands* with different flow configurations or plant species [58]. In addition, disinfection methods including chlorination, ultraviolet (UV) irradiation and sequential UV/chlorination treatment on the inactivation of ARGs have been studied [54, 59, 60]. Recently, nanomaterials with antimicrobial activity have been offered as a novel defense against ARGs [61]. Moreover, the removal of ARGs from treated wastewater in the coagulation process was examined [62]. In one of the recent works, the effect of biochar amendment on soil ARGs was assessed and the outcomes

Many diverse combinations of *nanomaterial* have proved that antimicrobial nanotechnology can be effective defenses against drug-resistant organisms, ARB, and ARGs. Two different mechanisms are probable when nanoparticles treat antibiotic resistance; the first mechanism is called Trojan Horse that develops drug-delivery characteristics. In this system, a functionalized nanomaterial is joined with antibiotics and nanomaterial enters inside cells and afterward discharge significant amounts of toxic ions [57]. In the second system, a mix of antibiotic and nanomaterials result in synergistic impacts, that means they battle ARGs independently [61]. Meanwhile, removal efficiency and mechanism of four ARGs including *tetA*, *sul2*, *ermB*, and *ampC* have been found using graphene oxide nanosheet. The removal efficiency was reported in the range of 2.88 to 3.11 logs at 300 µg/mL nanosheet solution showing the potential of graphene oxide nanosheet as an innovative and effective adsorbent for treatment of ARGs [64]. The potential for antimicrobial nanomaterials to restrict the propagation of multi-drug resistant pathogens while avoiding the generation of new nanomaterial-resistant organisms was studied by a group of researchers led by Aruguete [61]. They prepared a combination of nanomaterials functionalized with molecular antibiotics. This combination consisted of liposomes,

to 7.06-log in MBR [56].

load in wastewater [57].

sludge bioreactors in treatment plants.

showed that biochar is pretty operational [63].

Presence of specific genes encoding resistance to tetracyclines (*tetQ* [48]*, tetA* [49], and *tetO* [50]), sulfonamide (*sul*1 [49] *sul*2 [50]), erythromycin (*mphB* [49]), quinolone (*qnrD* [49] and *qnrS* [50]), beta-lactams (*cepA, cfxA* [48]*, blaCTX-M,* and *blaTEM* [50]), erythromycin (*ermB*), methicillin (*mecA*), vancomycin (*vanA)* [50], and aminoglycoside (*aac(3)-II, aacA4, aadA, aadB, aadE, aphA1, aphA2, strA* and *strB* [51]) were analyzed and confirmed by recent studies. The results of these studies prove that WWTPs are the main source of antibiotic resistance transmission.
